The Roles of Pteridine Reductase 1 and Dihydrofolate Reductase-Thymidylate Synthase in Pteridine Metabolism in the Protozoan Parasite Leishmania major

Abstract

Trypanosomatid protozoans depend upon exogenous sources of pteridines (pterins or folates) for growth. A broad spectrum pteridine reductase (PTR1) was recently identified in Leishmania major, whose sequence places it in the short chain alcohol dehydrogenase protein family although its enzymatic activities resemble dihydrofolate reductases. The properties of PTR1 suggested a role in essential pteridine salvage as well as in antifolate resistance. To prove this, we have characterized further the properties and relative roles of PTR1 and dihydrofolate reductase-thymidylate synthase in Leishmania pteridine metabolism, using purified enzymes and knockout mutants. Recombinant L. major and Leishmania tarentolae, and native L. major PTR1s, were tetramers of 30-kDa subunits and showed similar catalytic properties with pterins and folates (pH dependence, substrate inhibition with H 2 pteridines). Unlike PTR1, dihydrofolate reductase-thymidylate synthase showed weak activity with folate and no activity with pterins. Correspondingly, studies of ptr1 ؊ and dhfr-ts ؊ mutants implicated only PTR1 in the ability of L. major to grow on a wide array of pterins. PTR1 exhibited 2000-fold less sensitivity to inhibition by methotrexate than dihydrofolate reductase-thymidylate synthase, suggesting several mechanisms by which PTR1 may compromise antifolate inhibition in wild-type Leishmania and lines bearing PTR1 amplifications. We incorporate these results into a comprehensive model of pteridine metabolism and discuss its implications in chemotherapy of this important human pathogen.

Characterization of quinonoid-Dihydropteridine Reductase (QDPR) from the Lower Eukaryote Leishmania major

“…6,7-Dimethyl-5,6,7,8-tetrahydropterin (DMPH 4 ), NADPH, NADH, and horseradish peroxidase were purchased from Sigma. Folate-deficient medium was custom manufactured by Invitrogen and is identical to M199 except that it lacks folate and thymidine (18). All other reagents were of analytical grade.…”

“…Preparation of Crude Leishmania, T. brucei, and T. cruzi LysatesLeishmania promastigotes and T. brucei procyclics were collected by centrifugation at 1,250 ϫ g for 10 min at 4°C, washed twice with phosphate-buffered saline, and resuspended at 2 ϫ 10 9 cells/ml in 10 ml of Tris-Cl, pH 7.0, with 1 mM EDTA and a mixture of protease inhibitors as described (18). Frozen pellets of T. cruzi epimastigotes (Silvio strain) were generously provided by M. Pereira (Tufts University School of Medicine).…”

“…The importance of folate in essential metabolic processes such as synthesis of thymidylate has been established firmly in Leishmania by pharmacological and genetic studies both in vitro and in vivo (14 -16). Current data suggest that H 4 B is essential for growth in Leishmania (12,17,18) and plays a role in parasite virulence and differentiation (19). H 4 B has also been found to be a growth factor in Crithidia fasciculata and to effect proliferation and differentiation in various mammalian cell lines (20 -22).…”

Removal of Substrate Inhibition and Increase in Maximal Velocity in the Short Chain Dehydrogenase/Reductase Salutaridine Reductase Involved in Morphine Biosynthesis

“…Substrate inhibition is a common phenomenon in enzymes with a Ordered Bi Bi mechanism, as has been shown for a member of the SDR family, pteridine reductase (33,34). To investigate the mechanism of substrate inhibition we performed product inhibition studies with NADP ϩ , which inhibited SalR with a K i of 1.2 M (data not shown).…”

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

“…Methotrexate inhibits dihydrofolate reductase-thymidylate synthase (DHFR-TS) and to a much lesser extend pteridine reductase 43 . Comparison of 2DE maps of resistant and sensitive L. major parasite revealed a number of differentially expressed protein spots.…”